Various diseases display similar symptoms, but typically, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) diagnostics and more traditional forms of diagnostics only test for the presence of a single type of pathogen. Due to the prevalence of HCV in the US, we determined there was a need for a single diagnostic test that could differentiate between HCV subtypes, specifically 1a and 1b. We explored the feasibility of combining multiple Cas (CRISPR-associated protein) orthologs and Cas predecessors based on temperature of trans-cleavage activity, allowing for the detection of up to four targets, without the need for expensive equipment such as a thermocycler or multiple fluorescent channels. To limit cross-over activity and widen the threshold between positive and negative samples, we also incorporated RNA thermometers, such as HSP17, that could block cleavage activity at lower temperatures. With the increasing threat of emergent pathogens, we believe that the multiplexing of Cas orthologs for disease detection can have a broad impact on diagnostics for human health as well as diagnostics of plant and animal pathogens within the agricultural industry.
